1. Field of the Invention
The present invention relates to an apparatus and methods for the purification and/or isolation of biological samples. More specifically, the present invention relates to an apparatus and methods for electrically eluting and concentrating biological samples such as DNA or proteins separated on electrophoresis gels.
2. Description of the Prior Art
The use of gel electrophoresis to separate a heterogeneous mixture into constituent molecules is an effective and widely accepted method for assaying and/or analyzing biological samples. Nucleic acids, proteins, and other constituent biomolecules migrate in an unidirectional or bidirectional electrical field to bands or spots along a gel according to their electrophoretic properties. These properties include 1) charge, 2) molecular weight, and 3) structural configuration. Typically, these bands or spots are prepared for visualization using dyes applied to the gel after separation. Alternatively, markers or labels may be tagged to the constituent molecules of interest before separation. An overview of procedures for gel electrophoretic separation is given in RECOMBINANT DNA TECHNIQUES: AN INTRODUCTION, by Rodriguez and Tait, 1983 Benjamin/Cummings Pub., pp 67-79. Though a powerful tool for analysis, gel electrophoresis has up to now been of limited utility in the extraction of separated molecules due to the difficulty in removing the molecules from the gel in a pure concentrate. Various methods and devices are known for extracting nucleic acids and proteins from gel medium.
Wim Gaastra and Per Lina Jorgensen, "The Extraction and Isolation of DNA from Gels," Methods in Molecular Biology, vol. 2, 1984 Humana Press, pp 67-76, discuss standard protocols presently practiced in the art for extraction from gels. Of particular interest is the discussion of electroelution methods (Methods 1 and 2, pp. 69-71). Though these methods are disclosed to have the advantage of producing the highest yields of sample eluted from gels, electroelution procedures generally have disadvantages such as high expense, difficult handling, and/or loss of sample from dilution.
Other advantages and concomitant disadvantages of current techniques in electroelution of biological samples from gels are discussed in Current Protocols in Molecular Biology, 1993, Ausubel et al., editors, pp. 2.0.5-2.0.6, 2.6.1-2.6.2, and 10.5.1-10.5.2, as well as in "Gel Electrophoresis of DNA," Molecular Cloning, 1989, Sambrook et al, editors, pp. 6.2-6.23 and 6.28-6.29. From these discussions, it can be seen that the dialysis bag type electroelution method, with all its noted disadvantages, remains a standard practice.
In efforts to overcome some of the deficiencies in these standard protocols, modifications have been proposed, both in terms of procedural steps and in specialized equipment. For example, H. Hansen, et al. "Rapid and Simple Purification of PCR Products by Direct Band Elution During Agarose Gel Electrophoresis," BioTechniques, 1993, pp. 28-29 discloses a modification of the trough method of electroelution. Therein troughs or trap slots are cut in an agarose separation gel, without the placement of DEAE-cellulose paper or a dialysis membrane in the trap slot. Submarine electrophoresis is run in a unit with a UV-transparent bottom, for monitoring the band migration. Before the band of interest reaches the trap slot, the slot is filled with buffer, preferably of reduced pH to slow and focus the electrophoresis. Once the band containing the biological sample of interest has eluted into the trap slot, electrophoresis is halted, and the sample is removed from the trough with a micropipet. This method requires careful monitoring, as well as time-critical application and replenishment of a special buffer. Further, various considerations, such as the distance between loading and trap slots, UV illumination frequencies, and the like, require highly subjective yet critical decisions. Also, there is some loss of sample due to incomplete elution from the gel, reentrance of sample into the gel below the trap slot, and/or other physical limitations when the gel is not isolated from the collection area.
Zhen et al. in "A Simple and High Yield Method for Recovering DNA from Agarose Gels," Biotechniques, 1993, pp 894-898, disclose another trough elution method, wherein a band containing DNA of interest is cut from a preparatory separation gel, and placed in the cut trough of another gel, along with a polyethylene glycol (PEG) modified buffer. Electrophoresis is run until the DNA elutes out of the cut band and into the trough. The DNA is then pipetted into a microcentrifugation tube. The PEG additive must then be removed by extraction. This method suffers from the same limitations as the method of Hansen et al.
Various electroelution devices have been the subject of earlier patents. For example, U.S. Pat. No. 4,545,888 to Walsh discloses an electrophoresis device for the recovery of nucleic acids and other substances wherein large charged molecules are recovered from a separation gel to a binding cellulose resin through a plurality of transfer chambers. The use of the binding resin in this patent requires a further elution step, leading to loss of sample during transfer and elution.
U.S. Pat. Nos. 4,750,982 to Tomblin et al. and 4,863,582 to Wijanco et al. disclose devices for purifying and concentrating DNA onto a dialysis membrane. Though these devices include various segments of decreasing diameter, the dialysis membrane at the plane of contact with samples is flat. These devices are usually quite complicated, space consuming, and use duplicate elements already necessary for preparatory electrophoretic separation. Processes for using these devices are complicated and time consuming, leading to low recovery efficiency. Further, there is no sample concentrating effect during elution.
U.S. Pat. No. 4,964,961 to Brautigam et al. discloses devices useful for electroelution which feature a tapered tube, and a separable collection cup having a circular planar collection membrane. There is loss of sample when employing the device of Brautigam. Again, samples are not concentrated during elution. Similarly, U.S. Pat. No. 5,102,518 to Doering et al. discloses an electroelution device designed for use with epindorf centrifugation tubes. Bubble formation at the bottom of the collecting tube and the complicated processing procedures required decrease the recovery efficiency when employing this device.
None of the above inventions and patents, taken either singly or in combination, is seen to describe the instant invention as claimed.